Oksana Polesskaya

Human Immunodeficiency Virus-1 Tat Activates Calpain Proteases via the Ryanodine Receptor to Enhance Surface Dopamine Transporter Levels and Increase Transporter-Specific Uptake and Vmax

Human immunodeficiency virus-associated neurological disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy. The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, “reversible” DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging human immunodeficiency virus (HIV)-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinsons disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously, we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here, we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT Vmax, without changes in Km or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by total internal reflection fluorescence microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3β. We conclude that Tat activates RyRs via a calcium- and calpain-mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3β inhibition as clinical therapeutic approaches for HAND. Finally, we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients and by demonstrating similar findings in Tat-expressing transgenic mice.

Methamphetamine causes sustained depression in cerebral blood flow

The use prevalence of the highly addictive psychostimulant methamphetamine (MA) has been steadily increasing over the past decade. MA abuse has been associated with both transient and permanent alterations in cerebral blood flow (CBF), hemorrhage, cerebrovascular accidents and death. To understand MA-induced changes in CBF, we exposed C56BL/6 mice to an acute bolus of MA (5mg/kg MA, delivered IP). This elicited a biphasic CBF response, characterized by an initial transient increase (~ 5 minutes) followed by a prolonged decrease (~ 30 minutes) of approximately 25% relative to baseline CBF--as measured by laser Doppler flowmetry over the somatosensory cortex. To assess if this was due to catecholamine derived vasoconstriction, phentolamine, an α-adrenergic antagonist was administered prior to MA treatment. This reduced the initial increase in CBF but failed to prevent the subsequent, sustained decrease in CBF. Consistent with prior reports, MA caused a transient increase in mean arterial blood pressure, body temperature and respiratory rate. Elevated respiratory rate resulted in hypocapnia. When respiratory rate was controlled by artificially ventilating mice, blood PaCO(2) levels after MA exposure remained unchanged from physiologic levels, and the MA-induced decrease in CBF was abolished. In vivo two-photon imaging of cerebral blood vessels revealed sustained MA-induced vasoconstriction of pial arterioles, consistent with laser Doppler flowmetry data. These findings show that even a single, acute exposure to MA can result in profound changes in CBF, with potentially deleterious consequences for brain function.

Discovery, Synthesis, and Characterization of an Orally Bioavailable, Brain Penetrant Inhibitor of Mixed Lineage Kinase 3

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinsons disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production

Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorder (HAND) is likely neuroinflammatory in origin, believed to be triggered by inflammatory and oxidative stress responses to cytokines and HIV protein gene products such as the HIV transactivator of transcription (Tat). Here we demonstrate increased messenger RNA for nuclear factor-kappa B (NF-κB) family member, transcription factor RelB, in the brain of doxycycline-induced Tat transgenic mice, and increased RelB synthesis in Tat-exposed microglial cells. Since genetic ablation of RelB in mice leads to multi-organ inflammation, we hypothesized that Tat-induced, newly synthesized RelB inhibits cytokine production by microglial cells, possibly through the formation of transcriptionally inactive RelB/RelA complexes. Indeed, tumor necrosis factor-alpha (TNFα) production in monocytes isolated from RelB deficient mice was significantly higher than in monocytes isolated from RelB expressing controls. Moreover, RelB overexpression in microglial cells inhibited Tat-induced TNFα synthesis in a manner that involved transcriptional repression of the TNFα promoter, and increased phosphorylation of RelA at serine 276, a prerequisite for increased RelB/RelA protein interactions. The Rel-homology-domain within RelB was necessary for this interaction. Overexpression of RelA itself, in turn, significantly increased TNFα promoter activity, an effect that was completely blocked by RelB overexpression. We conclude that RelB regulates TNFα cytokine synthesis by competitive interference binding with RelA, which leads to downregulation of TNFα production. Moreover, because Tat activates both RelB and TNFα in microglia, and because Tat induces inflammatory TNFα synthesis via NF-κB, we posit that RelB serves as a cryoprotective, anti-inflammatory, counter-regulatory mechanism for pathogenic NF-κB activation. These findings identify a novel regulatory pathway for controlling HIV-induced microglial activation and cytokine production that may have important therapeutic implications for the management of HAND.

Rebuilding synaptic architecture in HIV-1 associated neurocognitive disease: A therapeutic strategy based on modulation of mixed lineage kinase

SummaryWork from our laboratories has validated mixed lineage kinase type 3 (MLK3) as an enzyme pathologically activated in the CNS by human immunodeficiency virus 1 (HIV-1) neurotoxins. In this review, we discuss MLK3 activation in the context of the neuropathogenesis of HIV-1 associated neurocognitive deficits (HAND). We use findings from the literature to substantiate the neuropathologic relevance of MLK3 to neurodegenerative disease, with an emphasis on Parkinson’s disease that shares a number of important phenotypic and neuropathologic characteristics with HAND. We discuss signal transduction pathways downstream from MLK3 activation, with an emphasis on their involvement in microglia and neurons in preclinical models of HAND. Finally, we make a case for pharmacologic intervention targeted at inhibition of MLK3 as a strategy to reverse HAND, in light of the fact that combination antiretroviral therapy, despite successfully managing systemic infection of HIV-1, has been largely unsuccessful in eradicating HAND.

MLK3 regulates fMLP-stimulated neutrophil motility.

INTRODUCTION Mixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP. METHODS The expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3-/- mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3-/- mice. RESULTS The expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3-/- mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3-/- mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3-/- mice-thereby confirming the role of MLK3 in neutrophil migration. CONCLUSIONS Mlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.

Transient hypercapnia reveals an underlying cerebrovascular pathology in a murine model for HIV-1 associated neuroinflammation: role of NO-cGMP signaling and normalization by inhibition of cyclic nucleotide phosphodiesterase-5

BackgroundCerebral blood flow (CBF) is known to be dysregulated in persons with human immunodeficiency virus 1 (HIV-1), for uncertain reasons. This is an important issue because impaired vasoreactivity has been associated with increased risk of ischemic stroke, elevated overall cardiovascular risk and cognitive impairment.MethodsTo test whether dysregulation of CBF might be due to virally-induced neuroinflammation, we used a well-defined animal model (GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic (Tat-tg) mice). We then exposed the mice to a brief hypercapnic stimulus, and assessed cerebrovascular reactivity by measuring 1) changes in cerebral blood flow, using laser Doppler flowmetry and 2) changes in vascular dilation, using in vivo two-photon imaging.ResultsExposure to brief hypercapnia revealed an underlying cerebrovascular pathology in Tat-tg mice. In control animals, brief hypercapnia induced a brisk increase in cortical flow (20.8% above baseline) and vascular dilation, as measured by laser Doppler flowmetry and in vivo two-photon microscopy. These responses were significantly attenuated in Tat-tg mice (11.6% above baseline), but cortical microvascular morphology and capillary density were unaltered, suggesting that the functional pathology was not secondary to vascular remodeling. To examine the mechanistic basis for the diminished cerebrovascular response to brief hypercapnia, Tat-tg mice were treated with 1) gisadenafil, a phosphodiesterase 5 (PDE5) inhibitor and 2) tetrahydrobiopterin (BH4). Gisadenafil largely restored the normal increase in cortical flow following hypercapnia in Tat-tg mice (17.5% above baseline), whereas BH4 had little effect. Gisadenafil also restored the dilation of small (<25 μm) arterioles following hypercapnia (19.1% versus 20.6% diameter increase in control and Tat-tg plus gisadenafil, respectively), although it failed to restore full dilation of larger (>25 μm) vessels.ConclusionsTaken together, these data show that HIV-associated neuroinflammation can cause cerebrovascular pathology through effects on cyclic guanosine monophosphate (cGMP) metabolism and possibly on PDE5 metabolism.

Pharmacologic Inhibition of MLK3 Kinase Activity Blocks the In Vitro Migratory Capacity of Breast Cancer Cells but Has No Effect on Breast Cancer Brain Metastasis in a Mouse Xenograft Model

Brain metastasis of breast cancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3) in controlling the in vitro migratory capacity of breast cancer cells, as well as the metastasis of MDA-MB-231 breast cancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasis of breast cancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration of breast cancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model of breast cancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size of breast cancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce the in vitro migratory capacity of breast cancer cells, but that it has no effect on either the frequency or size of breast cancer brain metastases, in a mouse xenograft model.

PCR-based detection of Pol III-transcribed transposons and its application to the rodent model of ultraviolet response

Cellular levels of RNAs containing transposable elements increase in response to various stresses. Polymerase III (Pol III)-dependent transcripts of transposons are different from transposon-containing RNAs generated by read-through Pol II-dependent transcription. Until now, Pol III transcripts were detected by primer extension followed by time-consuming gel electrophoresis. In this paper, we describe a more sensitive PCR-based method for the selective detection of Pol III-transcribed RNAs. The method is based on the difference in sequences at the 5′ ends of the Pol II- and Pol III-dependent transcripts. We employed this method to quantify Pol III transcripts of transposon B1 in rodent cells and revealed that their levels are affected by UV irradiation. We therefore conclude that the abundance of the Pol III-transcribed fraction of cellular RNA may serve as marker of stress response and can be conveniently quantified by the method described.

Chronic Central Nervous System Expression of HIV‐1 Tat Leads to Accelerated Rarefaction of Neocortical Capillaries and Loss of Red Blood Cell Velocity Heterogeneity

HIV‐1 infection of the CNS is associated with impairment of CBF and neurocognitive function, and accelerated signs of aging. As normal aging is associated with rarefaction of the cerebral vasculature, we set out to examine chronic viral effects on the cerebral vasculature.